REVIEW published: 30 June 2020 doi: 10.3389/fimmu.2020.01350

Cancer and HIV-1 Infection: Patterns of Chronic Antigen Exposure

Selena Vigano 1, Sara Bobisse 1, George Coukos 1, Matthieu Perreau 2 and Alexandre Harari 1*

1 Ludwig Institute for Cancer Research, University of Lausanne and Department of Oncology, University Hospital of Lausanne, Lausanne, Switzerland, 2 Service of Immunology and Allergy, University Hospital of Lausanne, Lausanne, Switzerland

The main role of the human immune system is to eliminate cells presenting foreign antigens and abnormal patterns, while maintaining self-tolerance. However, when facing highly variable pathogens or antigens very similar to self-antigens, this system can fail in completely eliminating the anomalies, leading to the establishment of chronic pathologies. Prototypical examples of immune system defeat are cancer and Human Immunodeficiency Virus-1 (HIV-1) infection. In both conditions, the immune system is persistently exposed to antigens leading to systemic inflammation, lack of generation of long-term memory and exhaustion of effector cells. This triggers a negative feedback loop where effector cells are unable to resolve the pathology and cannot be replaced due to the lack of a pool of undifferentiated, self-renewing memory T cells. In addition,

Edited by: in an attempt to reduce tissue damage due to chronic inflammation, antigen presenting Alejandra Pera, cells and myeloid components of the immune system activate systemic regulatory and Universidad de Córdoba, Spain tolerogenic programs. Beside these homologies shared between cancer and HIV-1 Reviewed by: infection, the immune system can be shaped differently depending on the type and Vijayakumar Velu, Emory University, United States distribution of the eliciting antigens with ultimate consequences at the phenotypic Tara Marlene Strutt, and functional level of immune exhaustion. T cell differentiation, functionality, cytotoxic University of Central Florida, United States potential and proliferation reserve, immune-cell polarization, upregulation of negative Christoph Neumann-Haefelin, regulators (immune checkpoint molecules) are indeed directly linked to the quantitative University of Freiburg, Germany and qualitative differences in priming and recalling conditions. Better understanding of *Correspondence: distinct mechanisms and functional consequences underlying disease-specific immune Alexandre Harari [email protected] cell dysfunction will contribute to further improve and personalize immunotherapy. In the present review, we describe relevant players of immune cell exhaustion in cancer Specialty section: and HIV-1 infection, and enumerate the best-defined hallmarks of T cell dysfunction. This article was submitted to Immunological Memory, Moreover, we highlight shared and divergent aspects of T cell exhaustion and T cell a section of the journal activation to the best of current knowledge. Frontiers in Immunology Keywords: HIV infection, cancer, lymphocytes, cellular immunity, exhaustion, senescence, anergy, immune Received: 09 February 2020 checkpoint Accepted: 27 May 2020 Published: 30 June 2020 Citation: INTRODUCTION Vigano S, Bobisse S, Coukos G, Perreau M and Harari A (2020) Cancer and HIV-1 Infection: Patterns of The primary function of the human immune system is to protect the host by reacting upon the Chronic Antigen Exposure. encounter of foreign antigens, as well as to prevent autoimmunity through self-recognition. Two Front. Immunol. 11:1350. arms orchestrate the activation of the immune system: the innate response triggered within the doi: 10.3389/fimmu.2020.01350 first hours and the adaptive response mounted over the following days, able to recognize and target

Frontiers in Immunology | www.frontiersin.org 1 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure specific antigens and to generate memory. T cells are the major EMERGENCE OF T CELL EXHAUSTION component of the adaptive immune system consisting of CD4 and CD8T cells (1), being the latter key players in the physical T cell exhaustion was initially described in the mouse model elimination of tumor and virus-infected cells. of LCMV infection (13–16), where, initially functional (17) Most naïve T cells encounter their targets, presented by and then transcriptional analyses led to the identification of professional antigen presenting cells (i.e., dendritic cells, DCs), PD-1 as first and main molecule associated with this status in secondary lymphoid organs (2). Such priming is crucial for (15, 18, 19). Afterwards, high PD-1 levels have been observed determining the acquisition of functional attributes by T cells in Simian Immunodeficiency Virus (SIV) infected Rhesus (3, 4). DCs govern the nature of primed T cells via the provision Macaques (15, 20–22) as well as in HIV-1 infected patients of processed antigens in the form of peptide/MHC complexes (23–25) and this was related to T cell impaired function and (signal I) and other important signals, including costimulatory disease progression. In HIV-1 infection, T cell exhaustion is interactions (signal II) and inflammatory cytokines (signal III) caused by antigen persistency and impaired CD4T cell help (5). Once activated, T cells undergo massive clonal expansion, (26, 27). During the acute phase of the infection, CD8 T cell differentiate into potent effectors, and express chemokines responses are generated, but they are incapable of mediating and homing receptors necessary for migration into peripheral complete virus clearance. HIV-1 is, indeed, endowed with a tissues. Effector CD4 T cells produce several cytokines depending high mutation rate capacity that leads to a quick and efficient on the polarization determined by the cognate antigen and escape from immune cells (28, 29). Moreover, lymphoid follicles the extracellular milieu, effector CD8 T cells express cytotoxic have been shown to be enriched in HIV-1/SIV-infected CD4 molecules, such as perforin and granzymes, and produce effector cells, and poorly infiltrated by CD8 T cells during early SIV cytokines. The production of cytotoxic molecules and cytokines infection. Consistently, the frequency of SIV-specific CD8 T cells is needed to help contain the spread of pathogens and tumors. entering the lymphoid follicles is inversely associated with the The fate of naïve CD8T cell differentiation is also determined levels of infected cells, suggesting a new mechanism of viral by interdependent variables such as frequency of contact with persistency (30). While infected cells are not eradicated, T the immunological synapses, epitope antigenicity, T cell receptor cells are continuously exposed to viral antigens, leading to a (TCR) affinity for cognate targets and the presence of CD4 T cell permanent expression of negative receptors and consequently to help (6). After CD8 T cell expansion and antigen elimination, T cell dysfunction (15, 31–34). Of note, beside antigen escape, any further immune activation is prevented by the upregulation HIV-1 preferentially infects HIV-1-specific CD4 T cells (35), and engagement of co-inhibitory molecules such as Cytotoxic leading to profound consequences in the immune-pathogenesis T Lymphocyte Antigen-4 (CTLA-4) and Programmed Death- of the disease (28). HIV-1-specific CD4 T cells expand at high 1 (PD-1). Most effector T cells die by apoptosis (contraction frequency during the early phase of the infection. Later on, phase), but about 5–10% survive and differentiate into memory their number decreases in blood and secondary lymphoid T cells. Different theories for memory T cell development have organs (36), due to killing by HIV-1-specific CD8T cells, virus been suggested (7), but recent findings strongly suggest that long- cytopathic effects and pyroptosis triggered by abortive viral lived memory CD8 T cells would arise from a subset of effector infection (37). In an early stage, CD8T cell responses are T cells through a process of dedifferentiation (8). Memory T also quickly impaired (27, 38–40), nevertheless this loss of cells are then maintained in the absence of antigens (homeostatic function is partially restored in the presence of HIV-1 specific expansion) and can exert rapid effector functions in response to CD4T cells (13, 27), highlighting the importance of CD4T cell previously encountered antigens (1, 9). depletion in determining CD8 T cell exhaustion. CD4 T cells Any disturbance of conventional activation signals may indeed provide help for CD8 T cells by producing supportive drive T lymphocytes to alternative cell fates, i.e., anergy, cytokines including interleukin (IL)-2 and IL-21, which can tolerance and exhaustion. This plasticity has evolved to act directly on the responding CD8 T cells (41–48). IL-2 has constrain autoimmunity and excessive immune responses a pivotal importance during priming of CD8 T cell response, that would otherwise cause undesired tissue damage and in order to generate functional memory cells able to perform immune-pathological conditions. Whereas, anergy is established homeostatic turnover and to mount potent secondary responses during priming, due to the absence of costimulatory signals, (49). IL-21 instead has a major role in sustaining and expanding and senescence is defined as growth arrest after extensive memory CD8 T cells (43, 44). In mice, CD4T cell help has been proliferation, exhausted T cells arise from cells which initially recapitulated by CD27 agonism that enhanced specific CD8T gained effector functions but became gradually dysfunctional cell effector functions in response to vaccination or a viral due to continuous TCR stimulation by persistent antigens infection (50). (10). Overlapping and discriminating functional and molecular Induction of T cell exhaustion is a common trait between features of these alternative cellular conditions have been HIV-1 infection and cancer (17), however key differences comprehensively investigated (11, 12). In the present review, we distinguish antiviral from anti-tumor immunity due to the describe the establishment and hallmarks of T cell exhaustion pathogenesis of the two diseases (Figure 1 and Table 1). The in HIV-1 infection and cancer. In addition, we highlight the immunogenicity of the tumor is shaped by the immune system parameters that allow the discrimination between functionally through a process called “immunoediting,” as the pivotal distinct T cell states, which are exhausted, activated, and memory work of Bob Schreiber first showed 15 years ago (116). In T cells. a first phase, the adaptive and the innate immune systems

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FIGURE 1 | CD8 T cell exhaustion in HIV-1 and cancer. T cell exhaustion in HIV-1 infection and cancer presents common origins and hallmarks, but also different features. The shared cause of T cell exhaustion is antigen persistency due to immune escape mechanisms. Moreover, in HIV-1 infection, the high viral mutational rate contributes to the immune escape while the preferential tropism of the virus for HIV-1 specific CD4 T cells leads to CD4 T cell loss that is also a main contributor of CD8 T cell exhaustion. In cancer, immunoediting and TME immunosuppression are peculiar determinants of tumor-specific CD8 T cell exhaustion. In both cancer and HIV-1 infection TOX has been identified as a master regulator of the transcriptional and epigenetic reprogramming of exhausted T cells. In HIV-1, T-betdim/Eomeshigh subset defines highly exhausted CD8 T cells, however in CD8 T cells from cancer patients T-bet and Eomes are expressed in T cells with different levels of exhaustion. At the protein level, the co-expression of many ICs has been identified as hallmark of T cell exhaustion in both cancer and HIV-1 infection. Exhausted cells are also characterized by functional and survival defects including reduced effector functions, expansion capacity and increased susceptibility to apoptosis. Metabolic rewiring is also a key player of T cell exhaustion, however in HIV-1-infected patients little/no information is available so far. synergize to recognize and eliminate malignant cells using rate and progressively undergo editing, resulting in tumor conventional mechanisms (elimination phase). These include: cell variants able to escape immune control (escape phase) the specific recognition of tumor-associated antigens and the (122). Clinically detectable tumors belong to this last and expression of effector molecules by T lymphocytes (type I most studied phase with cancer cells proliferating with no and II- interferon, perforin, Fas/FasL, tumor necrosis factor or limited constraints. Tumor cells directly induce T cell (TNF)-related apoptosis-inducing ligand—TRAIL), analogously exhaustion through the acquisition of somatic mutations, which to a viral infection, paralleled by the expression of recognition confer increased immune resistance and survival, altogether molecules such as NKG2D or ligands on tumor cells (induced contributing to prolonged antigen exposure. Ultimately, the by DNA damage and stress pathways) (117). Early infiltration of exhaustion state is the outcome of a transcriptional and tumors by immune cells such as pro-inflammatory macrophages, metabolic reprogramming induced by immunosuppressive both CD4 and CD8 T lymphocytes, NK, and DCs is crucial for cytokines (i.e., TGF-β, IL-10) and metabolites (i.e., lactate, tumor control (118–121). In the second phase, dormant tumor kynurenine, adenosine, PGE2) produced by cancer cells (121, cells survive in equilibrium with the immune system where 123) and tumor infiltrating immunosuppressive cell subsets, immunosuppressive and anti-tumor functions are balanced including regulatory T cells, myeloid-derived suppressor cells, (equilibrium phase). In this phase, the tumor microenvironment tumor-associated macrophages, cancer-associated fibroblasts, (TME) is composed of several cell types that produce variable adipocytes, and endothelial cells (124). Moreover, anti-tumor amounts of immune-suppressing and immune-stimulating T cells compete with cancer and immunosuppressive cells for molecules. In addition, tumors show a low proliferation nutrient availability and immunostimulatory factors. Current

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TABLE 1 | Hallmarks of exhaustion.

IC expression HIV-1/chronic infection Cancer PD-1 (23–25) PD-1 (51–56) CTLA-4 (57) CTLA-4 (53) TIM-3 (34, 58–60) TIM-3 (52, 53) LAG-3 (61) LAG-3 (53) TIGIT (62, 63) TIGIT (64–66) CD160 (33, 67, 68) CD160 (69) 2B4 (CD244) (67, 68) 2B4 (CD244) (70) BTLA (60) BTLA (71) CD6 (72) KLRG1 (73) VISTA (74–76) CD39 (77, 78) CXCL13 (53, 79) LAYN (80) Sia-SAMP:Siglec-9 (81) Transcription factors expressed by exhausted CD8 T cells HIV-1/chronic infection Cancer Master regulators: TOX, TCF-1 (82–85) EOMES 187 (85) STAT3 (86, 87) BLIMP-1 (88–91) BLIMP1 (55, 92) TOX (93) TOX (64, 93) NOTCH (94) NR4A2 (95) NFATc1 (96) NFAT (95) BATF (97–100) BATF (55) IRF4 (100) IRF4 (85) VHL (101) VHL (101) FOXO1 (102) FOXO1(102) PBX3 (19) FOXP1 (103) c-Myb (85) cMAF (104) GATA-3 (105) Zinc-dependent TFs (105) Epigenetic of exhaustion HIV-1/chronic infection Cancer PD-1 locus demethylation was observed in models of chronic infections (106) and in Tumor-reactive makers CD39 and CD103 are demethylated in tumor-reactive CD8 T HIV-1 infected patients (107) cells (whole-genome methylation profiling) (108) Increased accessibility to Pdcd, Havcr2, and Batf loci and to loci encoding genes Recurrence after anti-PD-1 therapy was associated with the hypermethylation of the involved in negative regulation of T cell effector functions(109) PD-L1 promoter (110) Recent studies show the stability of the PD-1 locus demethylation even after PD-1 Two chromatin states have been identified in exhausted T cells: (i) plastic and blockade (111) reversible, (ii) fixed dysfunctional state resistant to reprogramming (112) Identification of exhaustion-specific enhancer that contains essential RAR, T-bet, and HDAC6-selective inhibitors directed peripheral and infiltrating T cells toward a Sox3 motifs (109) Th1/effector phenotype (113) Exhausted T cells acquire heritable de novo methylation programs able to restrict T cell expansion and clonal diversity during PD-1 blockade treatment. A DNA-demethylating agent (Decitabine) improved T cell responses and tumor control during PD-1/PD-L1 blockade (114) 9–12 exhaustion clusters have been identified from epigenomic-guided mass cytometry profiling data (115)

data suggest that nutrient deprivation is inducing, per se, T cell depending upon the expression of PD-1, TIM-3, CD44, Eomes, dysfunction (125–127). T-bet, TCF-1, Slamf6, and CXCR5 (51, 67, 128–134). Exhausted CD8 T cell responses are quickly impaired during both early T cell progenitors were characterized in LCMV models as pool of viral infection and tumor establishment. cells expressing TCF-1+/PD-1int/CXCR5+/Slamf6+, responding Recently, terminally exhausted CD8 T cells have been to PD-1 blockade and differentiating into terminally exhausted characterized and distinguished from their progenitors CD8 T cells (TCF-1−/PD-1high/TIM-3+) (128–131). Their

Frontiers in Immunology | www.frontiersin.org 4 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure presence was also described among circulating tumor-reactive A cardinal feature of exhausted T cells in HIV-1 infection is the CD8T cells in melanoma patients and within TILs in primary sustained expression of multiple inhibitory immune checkpoints melanomas (135) and non-small-cell lung cancer (NSCLC) (ICs) (Table 1). (132). Interestingly, recent studies have better characterized The first and, to date, the most important IC involved in a subset of CD8+/CXCR5+ T cells with proliferative capacity CD8 T cell exhaustion in chronic infections (15, 23–25, 52, 158) and able to infiltrate B cell follicles and inflamed tissues in is PD-1. During chronic stimulation, PD-1 expression on virus- the presence of chronic antigen exposure and inflammation specific CD8T cells is high and sustained (23–25, 68) because (129, 131, 136–143). This subset shows heterogeneous phenotype of mechanisms involving both TFs [i.e., T-bet (159), Blimp-1 and gene expression profile depending on the pathogenic (88, 160)] and soluble factors [i.e., IFN-α (161) and RANTES context, still it is distinct from the CXCR5− counterpart pool and (156)]. In turn, PD-1 signaling affects the function, proliferation, maintain cytotoxic properties (144). In addition of being part of survival and chemotaxis of CD8T cells (23–25, 156, 162). In vivo, the TCF-1+/PD-1int progenitor pool (129, 145), these cells have PD-1high SIV-specific CD8T cells are characterized by a higher been described as having variable levels of exhaustion and being turnover (163). similar to Tfh cells (20, 108, 129, 131, 141, 146–149). This is The interaction of PD-1 with its two ligands PD-L1 and reflected in their capacity to help in the control of viral infection PD-L2 on hematopoietic and non-hematopoietic cells triggers and of tumor growth, in the promotion of inflammation and the phosphorylation of two cytoplasmic domains and the in the induction of B cell responses (108, 136, 137, 144). The subsequent recruitment of cytosolic tyrosine phosphatases formation and maintenance of the TCF-1+/PD-1int progenitor Shp2 and Shp1, the TCR-phosphorylating kinase Lck, and pool is orchestrated by the thymocyte selection-associated high the inhibitory tyrosine kinase Csk (164, 165). These effectors mobility group box protein TOX. While TOX is a key player mainly act by antagonizing the CD28 costimulatory signaling in the establishment of the exhausted state, its role is largely (166–168) and the TCR signaling via dephosphorylation of dispensable for the generation of effector and memory T cells. SLP76 and ZAP70 (164, 166). Moreover, signaling molecules Antigen persistency is likely to be the cause of Tox induction including ERK, Vav, PLCγ, PI3K, and Ras have been described since its expression is dependent on calcineurin and NFAT2. as downstream targets of PD-1 signaling in T cells, leading to TOX is therefore the translator of persistent stimulation into an impairment in metabolism, survival and cell growth (10, a distinct T cell transcriptional and epigenetic developmental 165, 168, 169). PD-1 is also expressed by CXCR5+ CD8 T cells program leading to T cell exhaustion. TOX is also important for (20, 170), a population particularly interesting for therapeutic the subsequent differentiation into terminally exhausted cells purposes. that is counteracted and regulated by the phosphatase PTPN2 In addition, landmark studies in LCMV (67) and then (82–84, 150–152). PTPN2 abrogation increases the number SIV/HIV-1 infection (33, 34, 62, 171, 172) highlighted the of terminally differentiated cytotoxic CD8 T cells promoting relevance of multiple ICs co-expression (i.e., CD160, 2B4, TIM- effective immune response, tumor/viral clearance and improved 3, T cell immunoreceptor with Ig and ITIM domains-TIGIT, response to inhibitory molecules blockade (84). TOX induces CTLA-4 and LAG-3) to define deeply exhausted virus-specific genes that are important for the exhaustion precursor formation, CD8 T cells. The co-expression of multiple ICs may be due to including transcription factors (TFs) (e.g., Tcf7, Nr4a2, and Tox their transcriptional co-regulation and non-redundant roles in itself) and co-inhibitory receptors (e.g., Pdcd1, Lag3, CD244, the physiological control of CD8 T cell responses (130, 173– and Havcr2). In conclusion, persistent activation and induction 176). Increased disease progression, viral replication and lower of TOX are common drivers of T cell exhaustion in both viral CD4 T cell counts were directly associated with PD-1 (23), infection and tumor pathogenesis. However, specific players CTLA-4 (171), TIM-3 (58, 59), LAG-3 (61), and TIGIT (62, such as CD4 T cell loss and TME heterogeneity in infection and 63) expression. In addition, the superior proliferative capacity cancer, respectively, contribute to define distinct and overlapping and the maintenance of cytotoxic functions by CXCR5+ CD8T traits of exhausted T cells in the two conditions. cells concur with a lower surface expression of ICs and a higher expression of co-stimulatory receptors (CD28 and ICOS) as opposed to the CXCR5− counterpart (129, 131, 148). Of HALLMARKS OF T CELL EXHAUSTION IN importance, SIV and HIV-1 specific CD8 T cell proliferation HIV-1 INFECTION in vitro improves when distinct ICs (i.e. CD160, 2B4, TIGIT, BTLA, TIM-3) are blocked (24, 33, 60, 62) and administration Many studies have indicated HIV-1-induced T cell exhaustion of anti-PD-1 in SIV infected macaques (177–181) and HIV- as main hallmark of the disease. Of note, HIV-1-specific CD8 T 1-infected patients (182) increases T cell immune responses, cells selectively show features of exhaustion as compared to however clinical efficacy remains controversial (181, 183– bulk CD8 T cell populations and unrelated virus-specific T cells 190). circulating in the same subject, as described in human and animal More recently, in SIV-infected macaques, the expression of studies (153–155). CD6 by PD-1+ CD8 T cells was associated with a reduced proliferation, cytokine secretion and cytotoxic capacity when Expression of Multiple ICs compared to their CD6− counterpart. The frequency of A complex network of stimulatory and inhibitory surface CD6+PD-1+ CD8 T cells positively correlated with SIV viral load molecules orchestrates the functionality of CD8 T cells (156, 157). and combined targeting of CD6 and PD-1 effectively restored the

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CD8 T cell proliferation capacity in vitro, suggesting that CD6 of TFs. Comprehensive whole-genome analysis of chromatin may be a new immunotherapeutic target (72). accessibility (ATAC-seq) (206), has shown that exhausted CD8 T Recently, the combination of transcriptomic and proteomic cells have a distinct epigenetic signature (95, 109, 111, 112, 207, data allowed the identification of multiple cell clusters that 208). For instance, exhausted CD8 T cells have several chromatin were evolving with HIV-1 disease progression or initiation regions with reduced accessibility (e.g., the Ifng, Ccr7, Il7r, Nt5e, of ART (64). These data may lead to the understanding of Tcf7, and Lef1 loci), while presenting open chromatin regions new specific features of disease evolution and drive novel in loci that govern the expression of IC molecules (e.g., Pdcd1, therapeutic approaches. Tigit, Ctla4), of ectoenzymes implicated in metabolic regulation (e.g., Cd38, Entpd1), of chemokines and cytokines (e.g., Xcl1) and of TFs (e.g., Eomes, Ikzf2, Tox)(64, 109, 111). The deletion Alteration in TFs Expression and of chromatin accessible regions including TF binding motif for Epigenetic Regulation RAR-retinoic acid receptor, T-bet, and Sox3 cause a dramatic Genomic approaches were recently applied to investigate the reduction in PD-1 expression, demonstrating their important transcriptional profile of virus-specific exhausted CD8 T cells, role in shaping exhausted T cell transcriptional profiling (64, 109, revealing their unique molecular signature as compared to non- 208). Moreover, during chronic LCMV infection, the Pdcd1 locus exhausted cells (Table 1)(19, 109, 111, 112, 115). Transcriptional become completely demethylated (106), while the histone H3 is analyses showed that exhaustion results from centrally connected less diacetylated in CD8T cells, indicating a loss in epigenetically pathways (19, 115, 191), having TOX as a master regulator. active genes (209). In parallel, the transcriptional regulatory Indeed, TOX expression correlates with the presence of an region of the PD-1 promoter is unmethylated in PD-1hi HIV-1- exhausted phenotype during chronic infections in mice (LCMV) specific CD8 T cells but not in donor-matched naive cells (PD- and humans (HCV) (82). In addition to TOX, several TFs 1−) (107). Thus, in chronic LCMV (106) and HIV-1 infection coordinate gene expression networks, including PBX3, EOMES, (107), PD-1 expression in virus-specific CD8 T cells is controlled BLIMP1 (Prdm1) (88–91), NOTCH (94), NFATc1 (96), basic by the chromatin accessibility of the gene itself (epigenetic leucine zipper transcription factor, ATF-like (BATF) (97–99), control) and by TF governing its expression (Figure 2). IRF-4, von Hippel–Lindau disease tumor suppressor (VHL), FOXO1, and FOXP1 (99–102, 130, 159, 192–198). At the Loss of Functions molecular level, TCR stimulation leads to the induction of Exhaustion in chronic viral infections has been described in both Tox expression (83) and induces the recruitment of TFs, like mice and humans as the progressive decrease in the capacity of Notch (94), NFATc-1 (96), IRF-4 and BATF (100), at the virus-specific CD8T cells to secrete cytokines, proliferate and promoter of different inhibitory receptors, ultimately driving exert cytotoxicity (23, 68, 210–213) as a consequence of persisting their upregulation. Among the genes induced by TOX, Tcf7 virus and antigen load (214). Loss of function characterizing (encoding TCF-1) promotes the generation of exhaustion exhaustion is hierarchical: IL-2 production is one of the first precursors through the induction of Eomes and c-Myb in early function to be extinguished, followed by TNF-α production, chronic infection, whereas PD-1 is needed to stabilize this pool whereas the ability to produce interferon-γ (IFN-γ) is more (85, 199). IRF4 was also shown to favor CD8 T cell exhaustion resistant to inactivation (155, 213, 215–218). while limiting memory T cell differentiation (100). Importantly, Consistently with the hierarchical loss of effector functions high high low high PD-1 /Eomes and PD-1 /T-bet T cells are both by exhausted T cells, Riley et al. (219) demonstrated that such necessary to contain chronic LCMV infection (130). However, effector functions depend on the strength of PD-1 signaling, thus dim high CD8 T cells presenting a T-bet /Eomes profile represent a on PD-1 expression levels. highly exhausted state with elevated levels of multiple inhibitory receptors (i.e., PD-1, CD160, and 2B4) (200, 201). In turn, PD- 1 signaling reduces the expression of Bcl-xl (168), favoring the HALLMARKS OF T CELL EXHAUSTION IN apoptosis of activated T cells (162, 202), and induces BATF CANCER leading to a decreased cytokine production, cytotoxic potential and proliferation rate of virus-specific CD8 T cells (97, 99). BATF The identification of exhausted T cells in the cancer setting induces the expression of T-bet and BLIMP-1 and correlates with was inspired by previous knowledge gained in chronic viral PD-1 expression in murine models of chronic viral infection. infections. In human melanoma metastasis, T cells sharing BLIMP-1 is upregulated in patients with progressive, as opposed many features of the exhaustion signature identified in LCMV to non-progressive, HIV-1 infection (194, 203, 204) and is also infection were found (53). However, as discussed above, the associated with reduced T cell proliferation and effector-cytokine establishment of exhaustion occurs differently in viral infection secretion capacity; however, these functions are restored by and cancer, the latter involving a complex network of players knocking down BATF or BLIMP-1 (88, 99). BLIMP-1 can also be and mediators. The repertoire of tumor-specific T lymphocytes induced in T cells upon priming with HIV-1 pulsed DCs together is generally devoid of highly avid autoreactive cells due to with other inhibitory molecules, including PD-1, TIM-3, LAG-3, central and peripheral tolerance mechanisms, and priming may and CTLA-4 (205). be inefficient due to the lack of co-stimulation, an inflammatory The characterization of the epigenetic landscape of exhausted milieu and/or the presence of immunoregulatory cellular subsets T cells gives novel and key insights to decipher the function (220). Therefore, a more heterogeneous pool of cells, fully

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FIGURE 2 | Epigenetic imprinting of T cell exhaustion. Shortly after antigen exposure, naïve T cells generate effector T cells that are armed to eliminate foreigner antigens. Activated T cells demethlyate loci dedicated to the expression of effector functions and activation genes. Activated genes include also ICs (i.e., PD-1) needed for starting the contraction phase once the antigen is cleared. After the contraction phase, memory T cells survive and present a specific transcriptional profile, while PD-1 expression is reduced. If the antigen persists, memory T cells cannot be generated and effector functions are progressively lost. In addition, some genes of the demethylated loci remain transcriptionally active sustaining the expression of ICs and leading to T cell functional exhaustion. In the context of a therapeutic intervention or physiological immune control of chronic antigen exposure (i.e., ART, Anti-PD-1, Elite controllers), exhausted T cells can restore, at least partially, their effector functions and reduce the expression of ICs such as PD-1. However, implicated loci remain demethylated, potentially causing a rapid restoration of the exhausted state after treatment interruption and the failure of the immune system to completely eradicate the antigens.

activated or not, may undergo the dysfunctional program. antigen exposure (18, 229). Exhausted T cells can co-express Consistently with their virus-specific counterparts, these cells PD-1 together with different ICs, including, LAG-3, CTLA- are characterized by increased expression of ICs (19, 64, 4, BTLA, TIGIT, 2B4 (CD244), VISTA, KLRG1 (53, 73, 230) 221), impaired homeostatic response to cytokines (222) and and TIM-3 (52, 131, 199). Inhibitory receptors signal through altered epigenetic and transcriptional programs (10, 191, 223). non-overlapping pathways and use different mechanisms to In contrast to HIV-1 infection where little/no information is regulate T cell function ultimately inducing exhaustion: they available to date, the rewiring of the T cell metabolism in sequester target receptors and ligands involved in activation cancer immunopathogenesis is a well-characterized hallmark of pathways (ectodomain competition), they dampen the signals exhaustion (224, 225). Of note, PD-L1 engaged by PD-1 acts from activating receptors and they mediate transcription of as an anti-apoptotic molecule and increases chemoresistance inhibitory genes (10). Importantly, the hierarchical co-expression on cancer cells through phosphorylation and activation of the of multiple inhibitory receptors has been associated with a more PI3K/AKT pathway, as opposed to inactivation in T cells (226– severe grade of cellular dysfunction (231). Additional, recently 228). Notwithstanding the recent burst of investigations on T cell identified markers of CD8 T cell exhaustion in cancer include: exhaustion in cancer, studies in human remain challenging and CD39 (77, 78), LAYN, whose expression is mutually exclusive animal models should be tuned to better reflect the slow course with LAG-3 in hepatocellular carcinoma patients (80), and of natural cancer progression and its antigenic contexts (high/low CXCL13 (53, 79). Moreover, Stanczak et al. (81) described the mutational load). Sia-SAMP:Siglec-9 as an inhibitory pathway in NSCLC, where high frequencies of Siglec9+CD8+ TILs inversely correlate with Expression of Multiple ICs survival (81). Finally, CD160+ CD8 T cells have been shown In line with what is described for HIV-1 infection, a high to express higher PD-1 levels than the CD160− counterpart, to and sustained expression of ICs is consensually considered as have less proliferative and cytotoxic potential and to be enriched the main hallmark of T cell exhaustion in the cancer setting among CD8 TILs in pancreatic cancer patients (69). Recently, a (Table 1). Tumor-specific CD8 TILs express high levels of PD- CXCR5+ CD8 T cell population has been observed to expand in 1 associated to impaired function (54). PD-1 is expressed diffuse large B cell lymphoma (232), follicular lymphoma (144) upon TCR engagement and NFAT nuclear translocation (96) and HBV-related hepatocellular carcinoma (137, 139, 141, 149). and may drive exhaustion of T cells undergoing persistent Circulating, tumor infiltrating, and lymphoid CXCR5+ CD8T

Frontiers in Immunology | www.frontiersin.org 7 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure cells were shown to co-express PD-1 and, in contrast with chronic (64, 115), those providing relevant insight for the design of IC viral infection (129, 131, 134, 148), TIM-3 (134, 140), however blockade therapies. they were functionally less exhausted than the CXCR5− CD8T cell population and expressed genes related to stem-like plasticity Loss of Functions and cytotoxicity (140, 141, 149). The frequency of this subset was As in chronic viral infections, exhausted T cells found in correlated with a better prognosis in follicular lymphoma (144), different tumor types have reduced effector functions as shown pancreatic (139), colorectal (137, 141), and lung (140) cancer, in terms of cytokine production and cytotoxicity (53, 237). suggesting its anti-tumor activity. However, combined blockade Nevertheless, the hierarchy by which T cells progressively lose of TIM-3, PD-1 or IL-10R pathways could increase the cytotoxic their functions is less clear (53, 54, 231, 237–239). TILs are activity of CXCR5+ CD8T cells indicating their limited lytic not functionally inert and, to some extent, contribute to tumor potential (139, 149). control (231, 240). The efficacy of IC inhibitors and IL-2-driven ex vivo expansion of functional TILs is an indirect proof of this impaired yet present anti-tumor activity. Furthermore, TILs Alteration in TFs Expression and can be highly heterogeneous among distinct cancer types as Epigenetic Regulation evidenced by their different capacity to respond to IC blockade. Tumor cells, together with immune and non-immune For instance, in small-cell lung cancer patients, subsets of PD- populations of the TME, contribute to a well-defined gene 1high TILs are enriched in tumor-specific T cells and their expression profile of dysfunctional anti-tumor T cells (Table 1), presence is a predictor of clinical response to anti-PD-1 therapy partially overlapping with that of exhausted T cells in chronic (132, 241–243). On the contrary, T cells infiltrating breast tumor infections, by releasing molecules and establishing inhibitory retain robust cytokine production and degranulation capacity contacts. In addition, recent studies in murine and human cancer (244) notwithstanding the expression of PD-1. In breast cancer suggest that TILs display a broad spectrum of dysfunctional states patients, PD-1 expression is therefore less predictive of TILs shaped by the multifaceted suppressive signals that occur within dysfunction and this may explain the modest clinical responses the TME (64, 130, 135). Several signaling pathways through the to anti-PD-1 or anti-PDL therapies. TCR, suppressive cytokines (TGF-ß, IL-6), inhibitory receptors, The proliferative potential of exhausted T cells is considered metabolites (adenosine, prostaglandins, lactate), enzymes (e.g., limited due to unresponsiveness to homeostatic cytokines such nitric oxide synthase, reactive oxygen species, indoleamine-2,3 as IL-7, IL-15 and IL-21 (211, 245, 246). However, the previously dioxygenase), low pH, hypoxia and nutrient deprivation, lead mentioned TCF-1+/ PD-1int progenitor pool of exhausted to the final transactivation of TFs controlling the expression of T cells has a residual proliferative potential that allows the different gene sets (101, 104, 173, 233, 234). As described for replenishment of the pool of exhausted antigen-specific CD8 T chronic infections, a complex pattern of TFs drives the initial cells by expanding and differentiating into the numerically triggering of differentiation toward the exhausted phenotype, larger population of TCF-1−/PD-1hi/TIM-3+ terminal progeny, including TOX, NFAT, Blimp-1, BATF, FoxO1, VHL, IRF4 characterized by a higher co-expression of other ICs and limited (93, 234), Bcl-6, cMAF, and STAT3 (86, 87, 104, 235, 236). proliferative capacity (135, 199). These factors exert distinct roles in T cells at different stages In a work by Li H. and co-workers, the intra-tumoral of differentiation and they do not exclusively govern gene immune infiltrates of 25 melanoma patients differing for expression in exhausted T cells. The epigenomes of different staging and treatments were analyzed by scRNA-seq for a T cell subsets contribute to the context-specific functions of deep characterization of dysfunctional T cells both in terms shared TFs. For instance, STAT3 dependent transcriptional of transcriptional states and TCR clonality (238). Exhausted T regulation limits both TILs recruitment and cytotoxic function cells expressing previously reported ICs (i.e., PD-1 and LAG- by downregulating IFN-γ, CXCR3, and CXCL10 expression 3) were observed in many patients. Importantly, intra-tumoral and inducing ROR-γt (87, 236). Of note, EOMES and T-bet CD8 T cells could cluster in two distinct subpools. T cells are expressed during the whole course of tumor progression belonging to the first pool spanned a wide range of transcriptional and, in contrast to chronic viral infections, they do not help in states, from transitioning to highly dysfunctional, expressed a distinguishing an exhausted-progenitor subset from terminally gradient of inhibitory molecules and were specifically observed differentiated exhausted T cells (Figure 1) (10, 55). More in tumor tissue. Some of the expressed regulatory molecules recently, new technological advances (i.e., mass cytometry (CSF1, ZBED2) were also shared with regulatory T cells. A second and single cell sequencing) are allowing a deeper examination subpool included T cells with cytotoxic potential, but limited of the molecular properties of dysfunctional T cells at the proliferative capacity. This second pool of T cells could represent single cell level. These studies represent milestones for the bystander T cells, likely from the circulation. Tumor-specific T comprehension of T cell biology in the context of complex cells were enriched in the exhausted pool, as previously observed TME, dominated by a high heterogeneity of cellular subsets. for NSCLC (132). Strikingly, T cells with an initial buildup of Recently, Bengsch et al. (64, 115) identified 9 distinct T cell the dysfunctional program maintained a clear proliferative signal clusters among exhausted CD8 T cells in HIV-1 infection and with a doubling time of few days and rapid turnover. This human lung cancer by using transcriptomic- and epigenetic- dynamic and active T cell state fits previously suggested models guided mass cytometry. This study also assigned an exhaustion of establishment of exhaustion at the tumor site (238). Common score to each of the subsets based on functional features mechanisms of the emergence of exhaustion are present among

Frontiers in Immunology | www.frontiersin.org 8 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure tumor types, but differences in the relative abundance of the As previously mentioned, the degree of exhaustion is directly subsets can be due to different TME, i.e., availability of antigens associated with the pattern of co-expression of different co- and exposure to inhibitory factors as well-shown for TILs in inhibitory receptors (67). First, this is mechanistically relevant, breast cancer (244). This is then reflected in the different capacity as simultaneous blocking of multiple ICs results in a synergistic to respond to IC blokade that is not only heterogeneous among reversal of T cell exhaustion in both cancer and chronic tumor types but also among individuals (244), as reviewed infections (171, 239, 261–263). Second, the identification of elsewhere (247). co-expression subsets may lead to a better discrimination In conclusion, in both HIV-1 infection and established between exhausted and activated T cells, reducing the risk for tumors, T cell exhaustion is likely driven by TOX and the off-targets effects. subsequent coordinated expression of several TFs. Exhausted Many studies have shown that chronic antigen stimulation T cells are characterized by loss of effector functions, high of T cells drives an IC expression pattern. For instance, TIM-3 expression of multiple ICs, reduced homeostatic expansion, and PD-1 cooperate for the induction of CD8 T cell exhaustion altered TFs expression, and remodeled chromatin. However, in cancer (52, 264–266) and chronic viral infections (34). In while in HIV-1 infection T-bet and EOMES allow the distinction LCMV infection, PD-1 and TIM-3 identify a population of T between progenitors and fully exhausted T cells, in cancer cells strongly enriched in gene signatures of terminal exhaustion patients TCF-1 and STAT3 may instead be the key TFs. The and harboring reduced proliferative capacity, longevity and avidity and the hierarchy of the loss of function of exhausted cytokine production (64). In HIV-1 infected patients, ART T cells in cancer patients is less well-described than in chronic significantly suppresses TIM-3 expression on HIV-1 specific infections. Exhausted cells present in the TME may be highly CD8T cells (267) indicating that, like PD-1, it is dependent heterogeneous and not include only the antigen-specific ones; on chronic TCR stimulation. Moreover, the expression profile new insights will explain how these aspects could affect the of CD56 and TIM-3 can discriminate between individuals that response to IC blockade. naturally control HIV-1 replication (elite patients) and ART- treated patients (268). After virus-clearance and CD4 T cell recovery, patients receiving ART show a quantitative loss of CD56+ CD8 T cells coupled to an exhausted phenotype, as shown EXHAUSTED VS. ACTIVATED/MEMORY by TIM-3 upregulation. Elite patients maintain a pool of cytolytic CD8 T CELLS CD56+ CD8 T cells comparable to healthy individuals. Similarly, CD160 expression also allows the distinction between exhausted Given the high heterogeneity and dynamicity of the memory and activated (PD-1+) HIV-1 specific CD8T cells (33). Indeed, CD8 T cell compartment (64, 238, 248), novel immunotherapies, only cells co-expressing CD160 and PD-1 (PD-1highCD160high) aiming at rescuing the functionality of exhausted T cells, would are functionally impaired in HIV-1 infected patients (33). require the ability to selectively distinguish exhausted from In cancer, the activation of ICs other than PD-1/PD-L1 memory and activated effector T cells. and CTLA-4 can be induced by adaptive resistance to IC therapies. The treatment of such tumors could benefit from the combination of anti-PD-1 with different immune checkpoint Expression of Surface Molecules molecules (e.g., LAG-3, TIM-3, TIGIT), activation markers The solely qualitative evaluation of ICs expression by CD8 T and cytokines/chemokines (269). On the other hand, T cell cells, per se, does not discriminate between exhausted and dysfunction is characterized by decreased levels of co-stimulatory activated T cells. As previously mentioned, inhibitory receptors molecules, of their ligands and of adaptor molecules impairing that are transiently expressed on activated effector T cells show the co-stimulatory signaling. Among these, CD44, LY6C, killer a higher and sustained upregulation on exhausted T cells, cell lectin-like receptor subfamily G member 1 (KLRG1), CD122 triggered by a persistent antigen stimulation. For instance, (IL-2Rβ), and CD127 (IL-7R), tumor necrosis factor receptor PD-1 is rapidly upregulated upon T cell activation (249) (TNFR)- associated factor 1 (TRAF1), CD28, and 41BBL have and persists at moderate levels in healthy subjects with a been described (67, 175, 246, 258, 259). In particular, exhausted preferential expression on effector memory T cells (162, 250– T cells display the same profile of effector T cells with reduced 253). During chronic infections, PD-1 expression on viral- telomere length and low levels of CD62L, CD127, and CD122 specific T cells increases (23, 38, 128, 254, 255) and does not expression (1, 40, 153, 215, 258, 270–273). Their incapacity to always reverse upon antigen removal (175, 256). HIV-1-infected respond to IL-7 and IL-15 (88, 128, 159, 245) lead to the lack patients responding to ART show reduced expression levels of homeostatic expansion in the absence of antigens (1, 88, 128, of PD-1 on virus-specific CD8 T cells after antigen clearance 130, 159) and, ultimately, to death (14, 154, 215, 256, 274–276). (257), still these levels are maintained above the physiological T cell dysfunction is also characterized by the downregulation of threshold observed in healthy individuals. This may be due to the signaling adaptor TNFR-associated factor 1 (TRAF1) both in a broad systemic immune activation, to the effects of common HIV-1 infected patients with progressive disease and in LCMV gamma-delta chain cytokines sustaining PD-1 expression on bulk chronically-infected mice (259). In HIV-1 infected patients, CD8T cells (258, 259) or to the irreversible transcriptional and TRAF1 expression negatively correlates with PD-1 expression epigenetic alteration affecting highly exhausted T cells (106, 260) and viral load and knockdown of TRAF1 in CD8 T cells from (Figure 2). viral controllers results in decreased HIV-1 suppression ex

Frontiers in Immunology | www.frontiersin.org 9 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure vivo. TGF-β is responsible for the post-translational loss of maintained upon metallothion-deletion, being uncoupled from TRAF1, while IL-7 signaling is able to restore TRAF1 levels. the gene dysfunctional module (105). Transfer of TRAF1+ memory T cells or a combination treatment Epigenetic studies also helped in identifying patterns with IL-7 and agonist anti-4-1BB antibody in chronic LCMV distinguishing T cell exhaustion from T cell activation/memory infection improve T cell expansion and viral control in a profile. Recent epigenetic studies in mice and humans indicate TRAF1-dependent manner (259). Patient samples of renal cell that exhausted T cells represent a unique T cell lineage, compared carcinoma also show reduced expression of TRAF1 compared to effector and memory T cells and are a stable, distinct and with normal kidney. This confers resistance to apoptosis and disease-relevant cell type (109, 111, 112). higher proliferative capacity to renal cancer cells (277). These HIV-1- and HCV-specific CD8 T cell genomes present a findings identify TRAF1 as a potential biomarker of T cell high accessibility to exhaustion-associated nucleotide regions. dysfunction and therapeutic target. Moreover, combining PD- On the opposite, the genome of CMV-specific CD8 T cells 1 blockade with an agonistic antibody to 4-1BB dramatically is characterized by a higher accessibility to memory-specific improved T cell function and LCMV control in vivo (278). nucleotide regions (109). Interestingly, the accessibility to Still, the role of positive co-stimulatory molecules in rescuing exhaustion-specific regions is reduced in CD8T cells specific exhausted T cells remains poorly described. for HCV epitopes that undergo viral escape (109), indicating that chronic exposure is needed to shape exhaustion-associated Transcriptional and Epigenetic Regulation epigenetic imprinting. Another key difference between exhausted and activated T cells Studies focusing on Pdcd1 locus revealed that during the resides in the TFs (18, 19, 191, 279). Both the quality of effector phase of an acute LCMV infection, the promoter the expressed TFs and the genes they can target, distinguish regions were largely demethylated to become remethylated as exhausted T cells from activated and memory CD8 T cells (191, the infection solved and CD8 T cell memory formed (106, 287). 280, 281). In the context of a chronic LCMV infection, the demethylation Transcriptional profiling analysis demonstrated that CD8 T observed in the Pdcd1 locus during chronic LCMV infection cell memory and exhaustion reflect distinct states defined by was instead stable and no remethylation was observed, even coordinated sets of modules. Specific genes and pathways when viral titers and PD-1 protein expression by exhausted differentially implicated in exhaustion vs. memory include genes CD8T cells decreased (106) or after transfer in recipient mice involved in epigenetics, DNA damage, and WNT signaling, such (260). Along the same lines, the unmethylated state of the Pdcd1 as Rtp4, Foxp1, Ikzf2, Zeb2, Lass6, Tox, and Eomes (191). The locus did not change in T cells from subjects with a viral load study by Bengsch et al. (64, 115) associates effector and exhausted controlled by ART for several years or from elite controllers T cells to a higher expression of CD39, LAG-3, TCF-1, Helios, (107). This suggests that the epigenetic program of the PD- CTLA-4 and PD-1, Eomes, TOX, 2B4, TIGIT, respectively. 1 locus is stabilized after prolonged exposure to HIV-1 virus During acute infection, T-bet and EOMES play pivotal roles in despite different levels of PD-1 surface expression. Consistently, the generation of terminally-differentiated (2, 282) and central- the transcriptome and the epigenome of terminally exhausted memory (283–285) CD8T cells respectively, while CD8 effector CD8 T cells (PD-1high/TCF-1−/TIM-3+) are stably rewired and T cells co-express T-bet and EOMES (286). In contrast, during resistant to remodeling after PD-1 blockade (111, 114)(Figure 2). chronic infection, exhausted T cell subsets express either T- These data strongly suggest that epigenetic remodeling may be bet or EOMES in a somehow mutually exclusive pattern and required to further improve strength and breadth of the efficacy they identify pools of non-terminal progenitor and terminally- of immune checkpoint blockade. differentiated exhausted CD8T cells, respectively (Figure 1) In conclusion, exhausted T cells can be distinguished from (130). Of note, anti-PDL1 therapy only improves the function activated T cells by the higher and sustained co-expression of of the T-bethi subset, while having little impact on EOMEShi IC molecules, as well as by a phenotype skewed toward effector cells (128, 130), indicating an important aspect of population memory cells with reduced co-stimulatory molecules expression. dynamics in IC blockade-mediated reversal of T cell exhaustion. Moreover, the systemically induced immune activation and A similar population of CD8 T cells responding to IC blockade, the stable transcriptional and epigenetic imprinting established (PD1int/TCF-1+), has been recently described as precursors during T cell exhaustion do not allow the restoration of of terminally exhausted cells (PD1high/TCF-1−/TIM-3+) to be IC molecules expression to the levels measured in healthy distinguished from memory precursors cells (PD1−/TCF-1+) on donors even after antigen removal/reduction. Among the TFs the basis of several epigenetic and transcriptional alterations such analyzed, T-bet, and EOMES allow the distinction between as higher expression of CXCR5 and Slamf6 (199). activated and exhausted CD8T cells in HIV-1 infection, By using a combined experimental and computational while metallothionins and GATA-3 have been suggested as approach, Singer et al. (105) described mutually exclusive gene discriminators in cancer patients. modules to distinguish dysfunctional from activated T cellsina murine colon carcinoma model. In particular, metallothionins, CONCLUSIONS responsible for regulating the intracellular zinc metabolism, and zinc-dependent TFs were found to be highly enriched in Recent major advances in immunotherapy ultimately dysfunctional CD8 TILs. GATA-3, a zinc-finger TF, consistently demonstrated the potentiality of the immune system in disease emerged as a driver of T cell dysfunction. Moreover, the control. However, they also proved that existing strategies are expression of the co-inhibitory receptors PD-1 and TIM-3 was hampered by the immune tolerance established by IC expression

Frontiers in Immunology | www.frontiersin.org 10 June 2020 | Volume 11 | Article 1350 Vigano et al. Patterns of Chronic Antigen Exposure on T cells. In addition, despite the significant difference in the AUTHOR CONTRIBUTIONS availability of clinical information concerning immunotherapy efficacy in cancer and HIV-1 infection, there is still a long way SV and SB wrote the manuscript. All authors contributed to the to go for the scientific community to decipher the mechanisms article and approved the submitted version. of immunosuppression in different indications. Recently, new technological advances (such as mass cytometry, single cell FUNDING sequencing, ATAseq, metabolomics) are allowing a deeper examination of the molecular properties of dysfunctional T cells This work was supported by the Swiss National Science at the single cell level. These studies represent milestones for Foundation (Grant 310030_182384). the comprehension of T cell biology in the context of complex TME, dominated by a high heterogeneity of cellular subsets and in HIV-1 infection where current immunotherapy may not ACKNOWLEDGMENTS improve T cell responses (64, 115). These data may lead to the understanding of new specific features of disease evolution and The authors acknowledge Mr. Samuel Cooper for his valuable drive novel immunotherapeutic approaches. proofreading of the manuscript.

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